PBMCs were isolated from heparinized blood by Ficoll-Hypaque density-gradient centrifugation (Pharmacia Biotech). In some experiments, cells were stimulated with 2.5 μg/ml anti-CD40 (eBioscience) and 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Calbiochem) at 37°C in an atmosphere of 5% CO2 for 48 hours. For stimulation with cytokines, 100 ng/ml recombinant human IL-6 (rhIL-6), 10 ng/ml rhTNF, 10 ng/ml rhIL-1β, or 100 ng/ml rhIL-17A (all from R&D Systems), each in combination with 2.5 μg/ml anti-CD40, was used. To assay for soluble RANKL in culture supernatants, B cells were isolated from PBMCs using CD19 microbeads, and separation was performed with a magnetic-activated cell sorter (Miltenyi Biotec). The cells were then cultured for 48 hours in the presence of anti-CD40 and PMA. Thereafter, the medium was replaced and the cells were returned to culture at 37°C in an atmosphere of 5% CO2 for an additional 5 days. Soluble RANKL was detected by enzyme-linked immunosorbent assay (ELISA; PeproTech), in accordance with the manufacturer’s protocol.
Rhtnf
Manufactured by R&D Systems
Sourced in United States
RhTNF is a recombinant human tumor necrosis factor alpha (TNF-α) protein. TNF-α is a cytokine that plays a central role in inflammation and the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 17a, by R&D Systems (2 mentions)
Magnetic activated cell sorter, by Miltenyi Biotec (1 mentions)
Recombinant human il 6 rhil 6, by R&D Systems (1 mentions)
Rhil 1β, by R&D Systems (1 mentions)
Anti cd40, by R&D Systems (1 mentions)
Anti cd40, by Thermo Fisher Scientific (1 mentions)
Pp242, by Merck Group (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Amino acid, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by PAN Biotech (1 mentions)
Recombinant human trail rhtrail, by R&D Systems (1 mentions)
Gam alexa 488, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Rhil 22, by R&D Systems (1 mentions)
Rhifn γ, by R&D Systems (1 mentions)
5 protocols using rhtnf
1
Isolation and Stimulation of PBMCs
2
Amino Acid Deprivation Impacts Rheumatoid Arthritis Fibroblast-Like Synoviocytes
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With approval of the local ethics committee, synovial tissues from patients fulfilling the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for RA (Aletaha et al., 2010 (link)) and patients suffering from OA were obtained as discarded specimens following synovectomy or joint replacement. Culture of FLSs was performed in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone) and with 1% penicillin and streptomycin (P/S) and nonessential amino acids (both Gibco) as previously described (Kiener et al., 2009 (link)). FLSs between passages 4 and 8 were used for all experiments. The following cytokines, inhibitors, and blocking antibody were used: rhTNF (R&D), JAK inhibitor I (Calbiochem), rapamycin (Calbiochem), MK2206 (Selleckchem), PP242 (Sigma), and Torin-1 (Tocris). All experiments were repeated with FLS cell lines from different donors.
To study the effects of amino acid deprivation, aa-free DMEM (Pan Biotech) was reconstituted with amino acids (Sigma) except the amino acid or amino acids to be omitted. The culture medium (DMEM ± aa) was changed 1 hr before TNF stimulation.
To study the effects of amino acid deprivation, aa-free DMEM (Pan Biotech) was reconstituted with amino acids (Sigma) except the amino acid or amino acids to be omitted. The culture medium (DMEM ± aa) was changed 1 hr before TNF stimulation.
3
Recombinant CD154 and CD95/Fas Signaling
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Recombinant soluble CD154 (sCD154) was generated as previously described [14 (link)]. Anti-CD95 or anti-Fas (clone CH-11, an IgM) was purchased from Chemicon (Temecula, CA). Recombinant human TRAIL (rhTRAIL) and rhTNF were obtained from R&D Systems (Minneapolis, USA). Anti-CD95 (LOB3/17, an IgG) was purchased from Bio-Rad Serotec Inc. (Raleigh, NC). The hybridoma producing antibodies raised against human CD154 (mAb C4.14) was produced in our laboratory. Anti-CD154 hybridoma 5C8 (IgG2a) and anti-CD40 hybridoma G28.5 (IgG1) were obtained from ATCC. Anti-αMβ2 antibodies (clone ICRF44) were obtained from BD Biosciences (Mississauga, ON). Anti-α5β1 (clone JBS5) and anti-αIIbβ3 (clone A2A9/6) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-β1 mAb (clones B44) was a generous gift from Dr. John A. Wilkins (Manitoba Centre for Proteomics and Rheumatic Diseases, University of Manitoba, Winnipeg, MB). The anti-TNF-R1 (clone H398), anti-TRAIL-R1 (clone 69036) and anti-TRAIL-R2 (clone 71908) antibodies were purchased from R&D Systems (Minneapolis, USA). The GAM-alexa488 was obtained from Invitrogen Life Technology (Burlington, Ontario, Canada). The biotin-labelled CD40-Fc was prepared using the method provided by Pierce (Rockford, IL, USA).
4
Neutralization of Soluble TNF via DN-TNF
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Recombinant human TNF (rhTNF; 12.5–100 ng/ml; R&D Systems; Minneapolis, MN) was used as a solTNF homologue. Dominant-negative TNF (DN-TNF; XPro1595/INB03; INmune Bio) is a next generation biologic that selectively neutralizes solTNF via quantitative exchange of individual subunits from the solTNF homotrimer [19 (link)]. For neutralization experiments, rhTNF was incubated in the presence of anti-human TNF antibody (R&D Systems; 10 µg/ml) or DN-TNF (10 µg/ml) in complete cell-culture medium at 37 °C for 1 h prior to cell treatment to ensure complete solTNF sequestration. Melanoma cell lines were cultured in 6-well plates (Corning, Manassas, VA) in the presence or absence of unscathed or pre-neutralized rhTNF for 48–72 h. Subsequently, melanomas were collected by gentle enzymatic dissociation using TrypLE Select (ThermoFisher Scientific; Pittsburgh, PA) and their phenotype and responsiveness to MAPKi were evaluated.
5
Keratinocyte Cytokine Response Assay
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Keratinocytes were isolated from skin biopsy specimens, as previously described. 21 Ten thousand keratinocytes at passage 3 or 4 were plated in Abbreviations used HBD2: Human b-defensin 2 MX1: Myxovirus 1 NLP: Never-lesional skin from patients with psoriasis pDC: Plasmacytoid dendritic cell rh: Recombinant human 48-well plates. At 100% confluence, cultures were exposed to recombinant human (rh) IL-17A (5 ng/mL), rhIL-22 (5 ng/mL), rhTNF (5 ng/mL), rhIFN-a (5 U/mL), or rhIFN-g (5 ng/mL) (all from R&D Systems, Minneapolis, Minn) for 16 hours.
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